Retroviral infections such as for example infections by the human immunodeficiency virus (HIV) are still one of the most important and most widespread human diseases.
One approach to treatment of retrovirus, e.g., HIV, is to target the provirus inserted into the genome of the host cell. Excision of the proviral DNA from the host's genome for example would prevent further HIV replication and differs from current methodologies in that it has the potential to eradicate even dormant virus present in the genome of the host.
One class of proteins that were considered for use in this alternative approach are site-specific recombinases (FLOWERS et al., 1997). Site-specific recombinases mediate a multitude of functions in nature from gene rearrangement to genome segregation, such as for example excision, inversion, or integration of defined DNA units (reviewed in STARK et al., 1992).
One of the simplest and best understood recombinases is the Cre recombinase from bacteriophage P1 that resolves genome dimers into monomers by recombination between two identical double-stranded DNA sites of a particular sequence (HOESS & ABREMSKI, 1985). The Cre recombinase has found widespread use in mouse genetics (NAGY, 2000). Cre is a 38 kDa protein that was named after is function, as it causes recombination (STERNBERG & HAMILTON, 1981). Prerequisite for this recombination is the alignment of two recombination sites recognised by Cre in antiparallel orientation which are then bound by four identical Cre subunits that join to form a ring in which each subunit contacts two adjacent subunits and one half site of one recombination site (HOESS & ABREMSKI, 1985). The recombination site recognised by Cre is a 34-bp double-stranded DNA sequence known as loxP (from locus of crossing over (x), P1; STERNBERG & HAMILTON, 1981), which is palindromic with the exception of its eight innermost base pairs (referred to as the spacer), which impart directionality to the site.
Some site-specific recombination systems, including the Cre/loxP-system function without accessory proteins or cofactors and function under a wide variety of cellular conditions. However, since the site-specific recombinases function through specific interactions of the recombinase enzyme subunits with their cognate DNA target sequences, the use of these enzymes is restricted by the requirement that the targeted DNA regions must contain appropriately positioned target sites (LEWANDOSKI, 2001). To date, no wild-type recombinase has been identified that recognises native retroviral sequences as their DNA target sequences.
Extensive mutational and structural analyses of site-specific recombinases have been carried out in recent years to alter their properties and to achieve a better understanding of the intricate mechanisms of these enzymes (for a review see VAN DUYNE, 2001; and COATES et al., 2005). A lot of studies focussed on the Cre recombinase to explore its evolvability. Several studies demonstrated that Cre target specificity could be altered when few nucleotides in its loxP recognition site were changed (BUCHHOLZ & STEWART, 2001; SANTORO & SCHULTZ, 2002; RUFER & SAUER, 2002). Further studies addresses the engineering of mutated loxP target sites containing sequences from the LTR of HIV-1 to develop possible target sites for the use of Cre as antiviral strategy (LEE & PARK, 1998; LEE et al., 2000).
The method of directed evolution is a powerful method to select enzymes with altered specificities (reviewed in Yuan et al., 2005; and JOHANNES & ZHAO, 2006). In the beginning this method was used to isolate improved enzymes on the basis of RNA by selecting RNA molecules with altered substrate sites. The use of PCR-based methods allows the screening of very large libraries and the recovery of successful coding regions from a pool of candidates. In the directed evolution of proteins, by contrast, the screening for and the recovery of improved mutants, which are identified by alterations in the properties of the protein, requires a method for retrieving the nucleic acid sequence encoding the protein. The link between the protein and its coding sequence has often been maintained by compartmentalisation. Consequently, library screening in directed protein evolution has been limited to “one-by-one” approaches that maintain the compartments, and the advantages associated with screening pools of candidates have not been available.
This limitation has been overcome by the development of methods that allow the crosslinking of proteins to their respective messenger RNAs (mRNAs) using mRNA-protein fusions and ribosome display. Functional screens for improved protein properties were thus coupled to direct retrieval of corresponding coding molecules, and large pools have been screened in vitro (see for example BUCHHOLZ et al., 1998). A further improvement of directed protein evolution was achieved by the so-called substrate-linked protein evolution (SLiPE; BUCHHOLZ & STEWART, 2001), wherein the substrate of the recombinase was placed on the same DNA molecule as the protein coding region. In this manner, when the recombinase was expressed within a compartment, its action altered the DNA substrate next to its own coding region. Consequently, a library could be screened as a pool by PCR to amplify only candidate coding regions that were next to an altered substrate. This allows the screening of large libraries conveniently for rapid retrieval of successful coding regions. This method was applied for altering the DNA specificity of Cre recombinase and adapting it to a new recognition target site (BUCHHOLZ & STEWART, 2001).
In view of the potential of site-specific recombinases and the need of finding an AIDS therapy eradicating HIV-1 provirus from the genome of host cell, WO 2008/083931 disclosed generation of a tailored recombinase (TRE) that is capable of recombining asymmetric target sites within the LTR of proviral DNA of a retrovirus inserted into the genome of a host cell, thus excising the provirus from the genome of the host cell. The engineered recombinase disclosed in the examples, Tre, recognizes a specific loxP-like site present in a particular HIV-1 strain. WO 2008/083931 appreciated that, due to the high sequence variability of retroviruses, in particular, HIV, for treatment of a patient with a different HIV strain, a different tailored recombinase might have to be adapted, or a collection of recombinases prepared containing tailored recombinases specific for a variety or target sequences.
In light of this, the inventors now addressed the problem of providing a tailored recombinase capable of excising a plurality of retrovirus, e.g., HIV strains. Thus, the generated recombinase can be employed for a plurality of HIV infections, without generation of a new recombinase for every strain. This problem is solved by the present invention.